A high percentage of the American workforce performs night shift work. These individuals have greater risk of fatigue-related occupational injuries, depression and chronic fatigue syndrome. Yet, little is known of the neural mechanisms underlying shift work sleepiness and fatigue. Circadian misalignment and sleep homeostasis contribute to sleepiness in shift workers, but may not fully explain symptomatology and consequences. We recently discovered that mice exposed to a sleep/wake pattern modeling three consecutive night shifts develop oxidative stress and hyperacetylation of mitochondrial proteins in locus coeruleus neurons (LCn) and partial loss of LCn, neurons essential for optimal alertness and brain health. Remarkably, mice re-exposed to this same sleep/wake pattern after a 4-day recovery period show increased oxidative stress and further LCn loss. We hypothesize that repeated exposures to sleep loss, as in intermittent night shift work, result in progressive metabolic dyshomeostasis and neuron loss in select wake-active neurons, including LCn, and that with frequent, repeated exposures to night shift sleep loss, irreversible wake impairments become evident. The proposed studies will determine the progressive nature of repeated exposures to shift work sleep disruption and then identify the molecular mechanisms underlying LCn injury with repeated sleep loss, modeling night shift work. To this end, we have identified potentially maladaptive responses to repeated sleep disruption in LCn involving sirtuins type 1 and 3 (SirT1 and SirT3). We find that SirT3 serves as an essential adaptive metabolic sensor and regulator in LCn in response to short-term wakefulness, yet this adaptive signal fails with repeated sleep loss, and LCn are lost. Previously we discovered that acute loss of brain SirT1 rapidly accelerates lipofuscin accumulation in LCn. Lipofuscin presents a source of irreversible oxidative stress. Now we find that repeated sleep loss depletes LCn SirT1 while increasing lipofuscin in LCn. Using unique conditional transgenic mice strains and gene therapy, we will examine the mechanisms underlying LCn injury in chronic intermittent sleep loss and determine the therapeutic potential for activating the Sirt1-SirT3 pathway in preventing neural injury and wake impairments in a model of shift work sleep loss.